New SARS-CoV-2 infection in a Pet Cat with severe lung disease in Italy

The pandemic respiratory disease COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan in December 2019 and then spread throughout the world; Italy was the most affected European country. Despite the close pet-human contact, little is known about the predisposition of pets to SARS-CoV-2. Among these, felines are the most susceptible. In this study, a domestic cat with clear symptoms of pneumonia, confirmed by Rx imaging, was found to be infected by SARS-CoV-2 using quantitative RT–qPCR from a nasal swab. This is the first Italian study reporting on the request of the scientific community to focus attention on the possible role of pets as a SARS-CoV-2 reservoir. An important question remains unanswered: did the cat die from SARS-CoV-2 infection?

The first epidemic cluster exploded in China, specifically in Wuhan city [2][3], as early as 1 December 2019. As of mid-July 2020, there have been over 5.525.245 confirmed COVID-19 cases worldwide, of which more than 30% cases in the EU and UK, with more than 347.108 global deaths [2] (https://www.worldometers.info/coronavirus. Accessed 10 May 2020). Specifically, Italy has been severely affected [4] and it was one of the first and hardest hit countries in Europe, with over 219.000 cases and 30.500 deaths reported [2], thus on 9 March 2020, a lockdown was declared for the entire country and progressively stricter restrictions were adopted [4,5].
The structure and the function of the SARS-CoV-2 are closely related to the already known coronaviruses responsible for the Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In detail, the mechanism of infection of the host cells is triggered by the external protein of the virus, the glycoprotein (S) peak, which is able to bind and recognize the angiotensin converting enzyme 2 (ACE2) of the human receptor, triggered by the serine of transmembrane protease type 2 (TMPRSS2), and/or extracellular matrix metalloproteinase inducer CD147 [2,[6][7][8].
Because of the close contact between people and pets, such as dogs and cats, the scientific community started investigating the possibility of animal-human virus transmission [9]. Furthermore, animal ACE2 receptor has high human ACE2 aminoacidic sequence identity [10][11][12]. It has been shown that some animal species, particularly felines, can occasionally of the disease. In addition, viral RNA was detected in 1:3 healthy cats exposed to infected felines, suggesting that they contracted the virus from the droplets exhaled by infected cats [13,19].
In this study we identified, for the first time, the natural infection of a cat by SARS-CoV-2 in Italy. The RNA extracted from the feline nasal swab was processed by RT-qPCR revealing the presence of two SARS-CoV-2 genes; moreover, a part of the gene was further sequenced to evaluate its nature. In addition, we excluded the presence of feline infectious peritonitis (FIP), a lethal systemic disease often associated with feline coronavirus (FCoV).

VETERINARY CLINICAL CASE
A sterilized male European shorthair cat was presented to a veterinary clinic by the owner reporting serious respiratory distress of about three days. On physical examination, the cat had severe dyspnea and sialorrhea, Kussmaul breathing, asynchronous chest and abdomen.
To confirm the suspicion of pneumoniae, a blood sample was collected from the jugular vein for hematological and biochemical analysis. Furthermore, given the similar symptomatology and the current situation caused by SARS-CoV-2, a nasal swab for SARS-CoV-2 was taken.
The main biochemical and hematological parameters revealed lower alkaline phosphatase and higher glycemia values than normal, while the blood count test revealed a relative and absolute neutrophilia (Figure 1a). Rx imaging revealed interstitial pneumonia with an area of pulmonary opacity (Figure 1b   Unlike several studies carried out on dogs [13], which have shown a low, if not absent, susceptibility to the virus, felines -especially cats -seem to have a greater predisposition to acquire the infection [13].
In the same way as other coronaviruses, such as SARS-CoV, the feline coronaviruses described until now seem to be able to switch their tropism, developing several specific virulent pathotypes that could even cause cross-species transmission [34]. Since the beginning of the pandemic, several cases of infected cats have been reported, although it is still not clear how the infection occurred and what type of symptoms is present [18].
In this study, we report -for the first time in Italy  sample alone is not enough to assert that it was the only cause of the pathological event [35].
However, it was likely one of the possible components responsible for progressive aggravation of the cat's health and its consequent death. To date, the crisis phase in some parts of the world seems to be over, but the natural reservoirs of the virus and its high contagion rate, as well as environmental and social conditions, could allow it to come back as a second wave. Particularly, as catsbut also pets and farm animals -are more common and in closer contact with humans than bats and seem to be a natural reservoir for the virus, they should be checked for SARS-CoV-2 when affected by severe pneumoniae. However, the question about the linkage between the SARS-CoV-2 infection and the cat death remain unsolved.

Specimen collection
A feline nasal swab was collected with the Virus Test Kit Diagnostics Sterile Pack Swabs Universal Viral Transport System (COD. RYCO-VART10B03, Jiangsu Rongye Technology Co., Ltd, Touqiao Town, Yangzhou City, China).

RNA Extraction
To increase the RNA uptake, a variation to the standard protocol was performed: 700 μl of swab buffer was processed in the same column, instead of the original 140 μl. AVE Buffer and ethanol were added at the same proportions in order to reach a final extraction volume of 6,3 ml. Then, the total volume was eluted in the same column 10 times. The rest of the extraction process was performed according to the protocol provided by the manufacturers.
Purification of viral RNA from the original swab was performed using QIAamp® Viral RNA minikit (COD. 52904 QIAGEN, Hilden, Germany).

RNA Quantification
Extracted RNA was quantified by the fluorimetric technique using the Qubit™️ RNA HS Assay Kit (Thermo Fisher, Catalog number: Q32852) according to the standard procedure.

RNA reverse transcription
Feline nasal swab RNA reverse transcription was carried out by using the QuantiTect® Reverse Transcription kit (QIAGEN, Hilden, Germany).